Linear polyacrylamide gel

A Freeman, Y Aharonowitz. Immobilization of microbial cells in crosslinked, polymerized linear polyacrylamide gels antibiotic production by immobilized Streptomyces clavuligerus cells. Biotechnol Bioeng 23 2747-2759, 1981. 

A major goal of the human genome project is to determine the order of occurrence of the four bases, adenine (A), cytosine (C), guanine (G), and thymine (T), in DNA molecules. The sequence defines an individual s genetic code. The need for sequencing DNA has spawned the development of several new analytical instruments. Among the most attractive of these approaches is capillary array electrophoresis. In this technique, as many as 96 capillaries are operated in parallel. The capillaries are filled with a separation matrix, normally a linear polyacrylamide gel. The capillaries have inner diameters of 35 to 75 p,m and are 30 to 60 cm in length. 

Changes in EM are caused by an increase of molecule size with unchanged charge. An increasing number of 2 -oxyethylene-substituted RNA-nucleotides in a dA[12]-mer leads to a linear increase (Fig. 24) of EMRe, which is due to the sieving effects of the linear polyacrylamide gel applied. Other substituents at the 2 -position give similar effects. However, with 2 -alkyl-subsituents the relation is not linear [149]. 

LDR products (44 and 51 bp) were analyzed by a cross-linked polyacrylamide gel (5%T/5%C) in approximately 45 min having a 1000-fold molar excess of the LDR primers (25 bp). Interestingly, when linear polyacrylamide gels were used, these same fragments could not be detected because of significant electrokinetic bias during the electrokinetic injection process. 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

Widhalm, A., Schwer, C., Blaas, D., Kenndler, E. (1991). Capillary zone electrophoresis with a linear, non-cross-linked polyacrylamide gel separation of proteins according to molecular mass. J. Chromatogr. 549, 446 451. 

The photograph (a) shows these proteins separated on a 5% polyacrylamide gel after treatment with 0.1% SDS. A plot (b) of log10RMM against the relative mobility of each protein shows a linear relationship and provides the basis for the determination of the relative molecular mass of an unknown protein. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

A, Absorption chi, chlorophyll car, carotenoid EET, excitonic energy transfer EF, exoplasmic fracture face EM, electron microscopy FWHM, full width at half maximum lEF, Isoelectric Focusing, LD, linear dichroism LHC, light harvesting complex PAGE, polyacrylamide gel electophoresis PF, protoplasmic fracture face PS, photosystem RC, reaction centre SDS, sodium dodecyl sulphate SSTT, single step transfer time. 

One of the most impressive examples for the detection of a transcription factor was published in 1996, when a mobility-shift assay in a linear-polyacrylamide-filled capillary using fluorescein-labeled DNA showed 100 times higher sensitivity than the conventional slab-gel technique with 32P. Furthermore, the detection of a transcription factor in a single sea urchin egg was demonstrated (24). 

EXAMPLE 12.5 Estimation of Number of Nucleotides in Glycine tRNA Using Electrophoresis. Synthetic DNA standards and RNA molecules were electrophoresed in 7 M urea solution on cross-linked polyacrylamide gels (Maniatis et al. 1975). A semilog plot of the number of nucleotides versus the mobility relative to xylene cyanol FF dye is linear and includes the points (N = 100, u i = 0.33) and (N = 50, urei = 0.55). Estimate the number of nucleotides in the glycine tRNA molecule of Staphylococcus epidermidis if it shows a relative mobility of 0.16. 

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